Intronic polymorphism (1541-1542delGT) of the constitutive heat shock protein 70 gene has functional significance and shows evidence of association with lung cancer risk

Somatic mutations of 11q23.3-linked constitutive heat shock protein 70 gene (HSPA8 alias HSC70) are detected by others in breast carcinomas. To examine whether intragenic, somatic mutations of HSPA8 occur in lung carcinomas, we sequenced its exons 2-8, with adjacent intronic sequences, in a series of DNA samples from non-small-cell lung cancers (NSCLC). Twenty-one polymorphisms were detected, but no somatic mutation. However, we observed an association between the HSC70 1541-1542delGT genotype and the immunohistochemical staining pattern of HSC70 protein. Tumors with weak (+) HSC70 protein staining were more frequent in the carriers of the polymorphic 1541-1542delGT allele than in the homozygotes of the major allele (20% vs. 6%, P=0.05 by Fisher's exact test). This statistically significant association prompted us to test the polymorphism functionally. The method we developed for the functional evaluation of intronic sequence alterations showed that the HSPA8 intron 2 with the deleted GT dinucleotide was associated with noticeable (approximately 20%) and statistically significant (P=0.005) reduction of the reporter gene activity. Our case-control analysis showed that the 1541-1542delGT heterozygous genotype was associated with significantly decreased risk for lung cancer (crude odds ratio (OR)=0.44; 95% confidence interval (CI): 0.23-0.84). To the best of our knowledge, this is the first report on the association between a polymorphism of a gene coding for the chaperone protein and lung cancer risk. Moreover, the simple method reported here, based on the dual-luciferase reporter assay system, can be useful for testing functional significance of polymorphisms located in introns of other genes.     

Expression and function of stress (heat shock) proteins in gastrointestinal tract

This review will focus on gastrointestinal mucosal protection against cytotoxic agents and cellular stress mainly from the viewpoint of expression and function of heat shock proteins, in their role of ‘molecular chaperones’, as internal cytoprotectants. Also, recently identified target molecules of heat shock protein in damaged gastric mucosal cells are introduced. Elucidation of such stress-responses and repairing process of damaged protein by heat shock proteins in the gastrointestinal mucosa may provide a better understanding for the mechanisms of cytoprotection and cellular repair. In addition, these findings in post-genomic level may provide new strategies for the therapy of gastrointestinal disorders.     

An immunoassay for heat shock protein 73/72: Use of the assay to correlate HSW3/72 levels in mammalian cells with heat response

An enzyme-linked immunosorbent assay (ELISA) for measurement of levels of heat shock proteins 73 and 72 (HSP73/72) in cultured cells and tissues is described. The assay involves detection of HSP73/72 in cell homogenates in 96-well plates using a specific monoclonal antibody. The assay has been used to explore the relationship between the amount of HSP73/72 in a cell and its response to heat shock, both before and after the development of thermotolerance. Six mammalian cell lines with differing responses to heat were characterized with respect to their response to heat treatments at 44°C and concentrations of HSP73/72. Contrary to the widely expressed idea that the amount of HSP73/72 dictates the degree of heat resistance, no positive correlation between levels of HSP73/72 and heat resistance was found for the six lines tested here: if one particular line, a mutant selected for heat resistance, was excluded from the analysis, there was a negative correlation between HSF'73/72 levels and heat resistance. A different result was, however, obtained when thermotolerant (transiently resistant) cells were compared to control cells. Here, we found a good correlation between the extent of thermotolerance and the amount of HSW3/72, suggesting that an increase in HSP73/72 level is important for the development of thermotolerance. The validity of the ELISA technique was checked using a second method for quantifying levels of HSP73/72. This involved uniform radiolabeling of cellular proteins, separation on two-dimensional gels and radioscanning to quantify radioactivity in each protein. The second technique is more powerful in that different isoforms of HSP73172 can be distinguished, but it is more difficult to perform, is more labour intensive and requires an expensive device for gel scanning. The results using the second technique agreed well with those from the immunoassay and indicated that the level of the highly inducible HSP72 correlated best with the extent of thermotolerance.     

非小细胞肺癌组织中热休克蛋白HSP70的表达及意义

目的:研究非小细胞肺癌(non-smallcelllungcancer,NSCLC)患者癌组织的热休克蛋白(heatshockprotein,HSP)的表达及病理学意义。方法:通过免疫组化的方法研究HSP70在NSCLC组织中的表达,分析其与NSCLC组织分型、分化程度及临床分期的关系。结果:47例NSCLC组织中37例HSP70表达阳性,较癌旁组织明显增加,P<0·01;26例鳞癌中20例为阳性表达,21例腺癌中17例阳性表达,P>0·05;16例高分化癌中有10例阳性表达,31例中低分化癌中27例表达阳性,P<0·05;20例Ⅰ~Ⅱ期癌中有13例阳性表达,27例Ⅲ~Ⅳ期癌中24例表达阳性,P<0·05;30例伴有淋巴结转移的NSCLC中有27例阳性表达,17例不伴有淋巴结转移的NSCLC中10例表达阳性,P<0·05。NSCLC组织HSP70表达显著升高,HSP70表达与NSCLC的临床分期相关,但与NSCLC组织类型无关。结论:HSP70在NSCLC的发展与演进中可能具有重要的意义。     

Activation of natural killer cells by heat shock protein 70

Intracellular heat shock proteins (HSP) function as molecular chaperones, they support folding and transport mechanisms of other proteins under physiological conditions and following physical or chemical stress. More recently, extracellular localized HSP have been found to play key roles in the induction of a cellular immune response. Either they act as carrier molecules for immunogenic peptides that are presented on Antigen Presenting Cells (APC) to cytotoxic T-cells or they themselves act as activatory molecules for the innate immune system. Binding of uncomplexed HSP to HSP-receptors on APC has been found to induce the secretion of inflammatory cytokines. Furthermore, an unusual tumor-selective membrane-localization of non-conserved regions of the 72 000 Da HSP (Hsp70) has been found to act as a recognition structure for natural killer (NK) cells. In this review the interaction of NK cells with Hsp70 or peptides derived thereof will be eluciated in more detail.     

Activation of natural killer cells by heat shock protein 70

Intracellular heat shock proteins (HSP) function as molecular chaperones, they support folding and transport mechanisms of other proteins under physiological conditions and following physical or chemical stress. More recently, extracellular localized HSP have been found to play key roles in the induction of a cellular immune response. Either they act as carrier molecules for immunogenic peptides that are presented on Antigen Presenting Cells (APC) to cytotoxic T-cells or they themselves act as activatory molecules for the innate immune system. Binding of uncomplexed HSP to HSP-receptors on APC has been found to induce the secretion of inflammatory cytokines. Furthermore, an unusual tumor-selective membrane-localization of non-conserved regions of the 72000Da HSP (Hsp70) has been found to act as a recognition structure for natural killer (NK) cells. In this review the interaction of NK cells with Hsp70 or peptides derived thereof will be eluciated in more detail.     

Heat shock cognate protein 70 encodes antigenic epitopes recognised by HLA-B4601-restricted cytotoxic T lymphocytes from cancer patients

Heat shock cognate protein 70 (HSC70), a highly conserved protein and a member of the family of molecular chaperones, has the ability to induce cytotoxic T lymphocyte (CTL) responses through binding and carrying antigenic peptides. We demonstrated in this study that the HSC70 gene encodes two antigenic peptides recognised by HLA-B46-restricted and tumour-reactive CTLs established from tumour-infiltrating lymphocytes of a colon cancer. These HSC70-derived peptides, at amino-acid positions 106-114 and 233-241, had the ability to induce HLA-B46-restricted and peptide-specific CTLs, which are reactive to tumour cells, from peripheral blood mononuclear cells of the majority of epithelial cancer patients tested. These results, along with those from the previous studies, indicate the two ways of HSC70 involvement in the immune responses to tumours: chaperones and antigens, and thus may provide a new insight for the development of HSC70-directed cancer-specific immunotherapy.     

小鼠肠癌HSP70多肽复合物的纯化及其抗肿瘤免疫效应

目的:应用AKTA-purifier100液相层析系统,建立分离和纯化热休克蛋白70(HSP70)多肽复合物的方法,并观察其抗肿瘤免疫保护效应。方法:采用DE-AE-Sepharose离子交换层析和ADP-Ag-arose亲和层析,从热处理的BALB/c鼠源性结肠癌细胞(CT26)制备裂解液中分离、纯化HSP70多肽复合物,并通过主动免疫保护实验观察其抗肿瘤免疫效应。结果:纯化蛋白经鉴定为相对分子质量70×103左右的HSP70多肽复合物;平均1g湿重肿瘤细胞可获得63.63μgHSP70多肽复合物;HSP70主动免疫的小鼠可抵抗CT26细胞的攻击。结论:用此层析法可分离纯度较高的HSP70,并可诱导明显的抗肿瘤免疫保护效应。     

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC.     

结肠癌中热休克蛋白70和糖调节蛋白94的表达与临床病理的关系

目的:探讨热休克蛋白70(HSP70)和糖调节蛋白94(grp94)在人结肠癌组织中的表达与临床病理的关系。方法:应用免疫组织化学和病理学图像分析方法研究80例人结肠癌组织及其癌旁组织、发生及未发生转移的癌组织中HSP70和grp94的表达。结果:在结肠癌组织中HSP70和grp94的表达明显高于癌旁组织(92.5%,85.0%VS56.3%,42.5%,P<0.01)。HSP70和grp94在中度和低度分化的结肠癌组织中的表达率明显高于癌旁组织(93.7%,87.5%;100%,90.0%VS56.3%,42.5%;P<0.01)。HSP70和grp94在DukesC期(97.1%,91.2%)和D分期(100%,90.9%)的表达比DukesA期(80.0%,70.0%)和B期(78.6%,71.4%)的癌组织阳性表达率高(P<0.05)。结论:HSP70和grp94在发生转移的低分化结肠癌组织和未发生转移的高分化癌组织中表达存在明显不同,它们的高表达可以作为结肠癌的诊断或预后指标。     

宫颈癌和宫颈上皮内瘤变组织中热休克蛋白70表达与人乳头瘤病毒感染的关系

目的　为了探讨宫颈癌和宫颈上皮内瘤变 (CIN )组织中热休克蛋白 70 (HSP70 )的表达与人乳头瘤病毒(HPV )感染的关系。方法　用免疫组化和原位PCR技术对 66例宫颈癌和 2 5例CIN组织进行检测。结果　宫颈癌、CIN和正常宫颈组织中HSP70表达率为 80 .3 %、72 .0 %和 2 0 .0 % (P <0 .0 1,P <0 .0 5 )。 5 2例HPVDNA阳性的宫颈癌组织中有 45例 (86.5 % )HSP70表达阳性 ,明显高于HPVDNA阴性组织 (5 7.1% ,P <0 .0 5 )。结论　HSP70与宫颈癌、CIN的发生发展有密切关系 ,HPV可以诱导宫颈癌和CIN细胞中HSP70的高表达。     

快速蛋白液相色谱系统分离与纯化小鼠结肠癌细胞热休克蛋白70

目的:应用快速蛋白液相色谱(fast protein liquid chromatography,FPLC)系统,建立分离和纯化BALB/c鼠源性结肠癌细胞(CT26)热休克蛋白70(heat shock protein70,HSP70)的方法。方法:用经43℃热处理12h的CT26细胞制备裂解液,经DEAE-Sepha-rose离子交换层析柱和ADP-Agarose亲和层析柱进行连续分离,所得蛋白经不连续SDS-聚丙烯酰胺电泳及Western-blotting进行蛋白相对分子质量及性质鉴定。结果:纯化蛋白经鉴定为相对分子质量70×103的HSP70。结论:通过FPLC系统可分离纯度较高的HSP70。     

人胃癌组织中热休克蛋白70和糖调节蛋白94的表达及其意义

目的：探讨休克蛋白70（HSP70）和糖调节蛋白94（grp94）在人胃癌组织中的表达与临床病理的关系。方法：应用免疫组织化学和病理学图像分析方法研究60例人胃癌组织及其癌旁组织、发生及未发生转移的癌组织中HSP70和grp94的表达。结果：在胃癌组织中HSP70和grp94的表达明显高于癌旁组织（93.3%,81.7%vs36.7%,25.0%,P〈0.01）。HSP70和grp94在低分化和发生转移的胃癌组织中的表达明显高于非转移癌和癌旁组织（90.0%,85.0%;100%,84.6%vs36.7%,25.0%;P〈0.01）。结论：HSP70和grp94在发生转移的低分化胃癌组织和未发生转移的高分化癌组织中表达存在明显不同,可以作为胃癌的诊断或预后指标。     

Photodynamic therapy and the role of heat shock protein 70

Photodynamic therapy (PDT) is an effective treatment for superficial epithelial skin cancers and is also being utilised for skin rejuvenation. PDT with porphyrin-derived photosensitisers may be capable of inducing rapid cytochrome c release initiating apoptotic cascade via an activation of different caspases. Hsp70 has been demonstrated to directly bind to the caspase recruitment domain (CARD) of apoptotic-protease activating factor 1 (Apaf-1), thereby preventing the recruitment of oligomerisation of Apaf-1 and association of Apaf-1 with procaspase 9. Further, cytoplasmic Hsp70 rapidly translocates to the cell surface where it stabilises damaged membranes and preserves their integrity depending on the PDT dose. The induced cell surface expression and release of Hsp70 and its relevance for tumor response or skin rejuvenation is not fully understood, but seems to be of interest for monitoring, predicting or optimising treatment regimens. This review aims to summarise the current knowledge on PDT mediated cell signalling.     

磷脂酶C-γ1在高温诱导热休克蛋白70表达中的作用

目的:研究磷脂酶 C -γ1(phospholipaseC -γ1,PLC- γ1)对高温诱导成纤维细胞热休克蛋白70(heat shock protein 70,HSP 70)表达的影响。方法:以基因敲除方法建立的缺失 plcg1 基因(plcg1 / )及野生型(plcg1+/+ )小鼠胚胎成纤维细胞为模型,Western 免疫印迹 增强化学发光法检测它们在高温诱导时的HSP 70表达,并以酶联免疫吸附( ELISA)法做半定量分析,MTT法对比分析两种细胞的热耐受力。结果:两种细胞经43℃与45℃高温诱导 30 min,都能引起HSP 70合成,但plcg1 / 细胞HSP 70表达水平显著低于 plcg1+/+ 细胞,P<0 01;较低温度(41℃)不能诱导 plcg1 / 细胞 HSP 70 表达,却能引起 plcg1+/+ 细胞 HSP - 70 表达,且 pl cg1 / 细胞的热耐力亦明显低于 plcg1+/+ 细胞。结论:PLC- γ1 参与了热诱导成纤维细胞 HSP70表达的信号转导。     

来源于人膀胱癌细胞株EJ的HSP70负载的DC所激发的特异性CTL细胞的体外应答效应

Objective: To investigate whether human dendritic cells (DC) derived from peripheral blood mononuclear cells (PBMC), which were pulsed by heat shock protein 70 (HSP70) isolated from human bladder tumor cell lines of EJ, were able to induce peptide specific cytotoxic T-lymphocytes (CTL) response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods: The EJ-derived HSP70 co-cultured with DC from the healthy volunteers' PBMC, along with the crude lysate (the supernatant before HSP70 purification) from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes (SI) and interferon-γ (IFN-γ) were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51Cr releasing test. Results: Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate (the supernatant before HSP70 purification) (P < 0.05). DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells (P < 0.05). Conclusion: The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.     

HSP70ASODN转染增强顺铂抑制肝癌细胞HepG2增殖的试验研究

目的:研究HSP70ASODN联合顺铂对于肝癌细胞的增殖抑制作用。方法:运用MTT法比较转染组和未转染组联合顺铂在肝癌细胞增殖方面的差异。结果:观察转染HSP70组与未转染组HepG2细胞株对化疗药物顺铂的敏感性时,24h、48h、72h、96h未转染组与转染组的细胞生长抑制率分别为,0.329±0.029、0.583±0.022;0.356±0.035、0.684±0.052;0.497±0.033、0.723±0.048;0.523±0.035、0.715±0.047。结论:HSP70ASODN能增强顺铂对HepG2增殖抑制作用。     

Enhancing the treatment effects of tumor cell purified autogenous heat shock protein 70-peptide complexes on HER-3-overexpressing breast cancer

Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes (HSP70-PCs) on HER-3-overexpressing breast cancer. Methods In this study, we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics. We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells (DCs) and CD8+ T cells induced by these complexes. Third, recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3–overexpressing breast cancer cells, and the resulting immunological response was examined. Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+ T cell killing of HER-3-positive breast cancer cells. Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.     

热休克蛋白70在乳腺癌中的表达及其与预后的关系

目的 探讨热休克蛋白70(heat shock protein70，HSP70)在乳腺癌中的表达，评估其在乳腺癌预后中的作用。方法 用免疫组化方法检测62例手术切除的乳腺癌组织中HSP70的表达，研究其与临床病理特征的关系及对预后的影响。结果 (1)HSP70在乳腺癌组织中的阳性表达率为56．5％(35／62)。(2)HSP70表达与肿瘤大小(P=0．021)及腋窝淋巴结转移(P=0．015)存在相关性，而与月经状况、组织学分级之间均无相关性。(3)HSP70阴性表达组的无病生存期(DFS)及总生存期(OS)均明显优于阳性表达组。(4)(20x回归单因素分析显示腋窝淋巴结转移个数、组织学分级、肿瘤大小、TNM分期及HSP70阳性表达与DFS及OS明显相关；Cox回归多因素分析表明仅有腋窝淋巴结转移个数、组织学分级及肿瘤大小与DFS及OS明显相关，而TNM分期及HSP70阳性表达进入Cox回归模型。结论 HSP70在乳腺癌组织中有一定程度的表达，HSP70阳性表达与乳腺癌患者生存期的缩短有关，由于多因素回归分析未能显示HSP70表达与DFS和OS相关，因此HSP70不能作为乳腺癌的独立的预后因素。     

Co-overexpression of Bag-1 and heat shock protein 70 in human epidermal squamous cell carcinoma: Bag-1-mediated resistance to 5-fluorouracil-induced apoptosis

Background:

The aim was to determine whether Bcl-2-associated athanogene-1 (Bag-1) and/or its binding protein heat shock protein-70 (Hsp70) exhibit deregulated expression in epidermal squamous cell carcinoma (SCC) and whether Bag-1 confers apoptosis resistance.

Method:

Immunohistochemistry for Bag-1 and Hsp70 was performed on 60 epidermal SCC and 10 normal skin samples. The epidermal SCC cell line SCC-13 was treated with 5-fluorouracil (5-FU) after Bag-1 knockdown to determine whether high Bag-1 levels contribute to growth and/or apoptosis resistance.

Results:

Normal epithelium expressed primarily nuclear Bag-1. Most tumours showed reduced nuclear Bag-1 staining, but a subset exhibited strong Bag-1 staining, with cytoplasmic Bag-1 staining intensity correlating with cytoplasmic Hsp70 staining intensity (r_s=0.462; P<0.001) and less differentiation (P<0.001). Bag-1 knockdown resulted in markedly reduced SCC-13 cell yield, increased spontaneous apoptosis and enhanced sensitivity to 5-FU-induced apoptosis. Apoptosis induced by 5-FU in the Bag-1-knockdown cells was significantly greater than the additive apoptotic effect of 5-FU or Bag-1 knockdown alone.

Conclusions:

Overexpression of Bag-1 and Hsp70 in poorly differentiated SCC may confer both enhanced tumour cell growth and apoptosis resistance. Bag-1 may contribute to the resistance of more advanced epidermal SCC to chemotherapy.